Hypericum Perforatum L. Oleoresin, Procedure for Obtaining it and Uses of it

ABSTRACT

Hypericum Perforatum L. Oleoresin, procedure for obtaining it and uses of it. A Hypericum Perforatum L Oleoresin is describes that is stable over time on its Hyperforin and Hypericine content without adding preservatives; a procedure for obtaining the Oleoresin by extraction with low-polarity solvents, followed by purification; also its use as a regulator of the components of the extracellular matrix is described and its use for the manufacture of hydrosoluble gels containing Hypericum Perforatum L Oleoresin.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention describes an oleoresin or lipidic extractof Hypericum perforatum L., which contains hypericine and is enrichedwith hyperforins, and is stable over time without adding stabilisers.The extract is obtained by extraction with low-polarity solvents andthen purification by re-extraction with alcanols-water. The inventiondescribes the use of the oleoresin to regulate the components of theextracellular matrix in a dose dependent manner.

[0002] The invention also relates to hydrosoluble gels, the activeingredient of which is Hypericum perforatum L., to be used ascicatrizants.

BACKGROUND OF THE INVENTION

[0003] Hypericum perforatum L has long been used by popular medicine, asa cicatrizant and in the treatment of burns.

[0004] The chemical species present in Hypericum perforatum L includeacilfloroglucinols: hyperforins and adhyperforins; Hypericum perforatumalso contains naptodiantrones: hypericine and pseudohypericine. Thesecompounds are responsible for the activity of the extracts in thetreatment of wounds and scars, but these products are unstable whenattempting to obtain them in pure form, and over time they decompose dueto the effect of light and heat.

[0005] European patent EP0854726 describes obtaining stable Hypericumperforatum L extracts by adding antioxidant preservatives such asascorbic acid, cysteine and/or glutathione, extracting the plant withorganic solvents or alcohol-water mixes.

[0006] On the other hand, the pharmaceutical formulations described instate of the art DE2406452 are ointments containing an active ingredientconsisting of fresh Hypericum leaves and oily excipients (olive oil,bees wax, fatty acid esters, etc.), providing a lipophylic ointment thatis insoluble in water.

[0007] However, these oily formulations lead to a maceration of the skinafter prolonged use, and have a reduced bioavailability making itdifficult to wash the lesions, and therefore to monitor their condition.

[0008] As mentioned earlier, Hypericum perforatum L extracts have showndifferent pharmacological activities, mainly as cicatrizants, but todate their effect on the modulation of the extracellular matrix (ECM) ofthe fibroblasts. The collagen components are therefore responsible forthe mechanical properties of the skin, whereas tenascine is responsiblefor the regulation of the adhesion molecules and migration incicatrisation processes.

[0009] The technical problem of the invention is providing stabilisedextracts of Hypericum perforatum L., without adding stablepreservatives, without losing its active ingredients and preserving itspharmacological properties. Moreover, its use in a hydrosoluble andstable pharmaceutical composition containing Hypericum perforatum Lextract enriched in hyperforins to avoid the maceration of the skin thattakes place with treatment with lipophylic ointments and creams,improving bioavailability and the evaluation of the lesions.

SUBJECT OF THE INVENTION

[0010] Surprisingly, the invention is bases on the stability of thelipidic extracts of Hypericum perforatum L., which contain all itsnatural components: hyperforins and hypercins, in a lipidic matrix, andare stable over time without adding preservatives. The lipidic extractsor oleoresins are obtained with low-polarity solvents capable ofextracting the lipidic components of the drug, followed by purificationby re-extraction with alcanols-water.

[0011] The extracts have more than 10% content in hyperforins and morethan 0.5% in hypericines, in a lipidic matrix that stabilises the activeingredients of Hypericum perforatum L.

[0012] The extracts have shown a dose-dependent activity in theregulation of the production of the components of the extracellularmatrix, thus avoiding the possible toxicity of the product by inhibitingthe production of collagen and tenascine at high doses and, on the otherhand, avoiding the formation of hypertrophic and cheloid scars.

[0013] The aforementioned extracts can be used in pharmaceuticalpreparations such as hydrosoluble gels as cicatrizant agents thatimprove bioavailability, prevent the maceration of the skin, and improvethe application of the product compared with the formulations describedby the state of the art based on oily or lipophylic excipients.Moreover, with hydrosoluble gels it is possible to better monitor thelesions, since they are transparent.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The invention is based on the stability of Hypericum perforatum Lextracts, characterised by the pressence of hypericine and hyperforinsin a lipidic matrix.

[0015] The extracts are obtained first by the extraction of Hypericumperforatum L with a low-polarity solvent, followed by re-extraction withhot alcohol-water mixes, obtaining a fluid oleoresin with over 10%content in hyperforins and more than 0.5% content in hypericines.

[0016] The fluid Hypericum perforatum L oleoresins obtained byextraction with low-polarity solvents, followed by hot mixes of alcanolswith low molecular weight and water, have been seen to be stable,without losing their hyperforin content due to the effect of light andtemperature. The hyperforin content after one year of storage at 40° C.,exposed to the light and room temperature, has shown a hyperforincontent of 15%.

[0017] In a preferred embodiment of the invention to prepare theextract, the Hypericum perforatum L is extracted at a low temperaturewith solvents with a polarity below 0.6. The solvent to be used is notcritical, and different mixes can be employed.

[0018] The plant is extracted in the proportion of one part drug to 6parts solvent, by maceration for 24 hours at a temperature less than orequal to 20° C. It is then filtered and the drug is extracted again bymaceration for 24 hours, and so on until extraction is complete.

[0019] The extracts in liquid form are concentrated to obtain a blandand fluid syrup, by high vacuum and a temperature below 40° C.

[0020] The bland fluid syrup is purified by dissolution in analcanols-water mix and filtered, preferably using alcohols of lowmolecular weight, such as methanol, ethanol or isopropanol. The solutionis prepared at 40-50° C., filtered and concentrated at reduced pressure,obtaining a fluid oleoresin.

[0021] With this extraction procedure, the oleoresins obtained have ahigh content in hyperforins of 10-15% and a content in hypericines of0.5%, stable over time, determined by chromatographic orspectrophotometric techniques. In order to optimise the extractionprocedure, additional steps can be added, such as selecting the initialraw materials with a hyperforin content of over 2%, dehydration of theraw materials at a temperature below 35° C., cryogenisation of theplant, etc.

[0022] On the other hand, according to the invention, hydrosoluble gelsare obtained that facilitate the release of liposoluble Hypericumperforatum L oleoresin, permitting diffusion between the structures ofthe corneal layer, obtaining more bioavailability than the oilysolutions. These hydrosoluble gels contain dilutant, humidifying,gelifying, emulsifying and preservative agents.

[0023] In one way of embodying the invention, the preservatives aredissolved in water and the Hypericum perforatum L oleoresin is dissolvedin the emulsifiers. This mix is added to the preservatives dissolved inwater, followed by the slow addition of the humidifying and gelifyingagents, avoiding the occlusion of air.

[0024] preferably water is used as a dilutant, glycerine as ahumidifier, glyceril palmitate as a gelifier, parabenes as preservativesand PEG-40-hydrogenated Castor Oil, Polysorbate-20 and Octoxinole-11 asemulsifiers.

[0025] The preferred proportions for the invention are as follows:Hypericum oleoresin 0.1-0.5% Water   60-80% Glycerine and glycerilPolyacrilate   20-30% PEG-40-Hydrogenated castor oil, Polysorbate-20 yOctoxinole-11    1-3% Parabenes 0.2-0.4%

[0026] Hypericum oleoresins regulate the production of the components ofthe extracellular matrix (ECM) such as collagen and tenastin, butsurprisingly, this regulation is dose-dependent.

[0027] Hypericum perforatum L oleoresins at low concentrations (0.5-1μg/ml) increase the production by 70% of the synthesis of collagens infibroblast cultures, but at concentrations greater than 5 μg/ml,collagen synthesis is inhibited, avoiding the possible toxicity of theproduct and avoiding the formation of hypertrophic or cheloid scars.

[0028] According to the results obtained in the invention, theproduction of tenascine decreases after the treatment of the fibroblastswith Hypericum oleoresin for 24 h at 37° C. in a dose-dependent manner.

[0029] Following is a description of the invention using characteristicexamples, to which the scope of the invention is not limited.

EXAMPLE 1 Illustrating the Procedure for Obtaining Oleoresins fromHypericum Perforatum L

[0030] 100 kilos of dehydrated Hypericum perforatum L flowers andleaves, at less than 35° C. and with a hyperforin content of greaterthan 2%, are macerated with 600 litres of methylene chloride: acetone(50:50) for 24 hours at 20° C. This process is repeated 3 more timesuntil extraction is complete. The liquid extracts are concentrated in avacuum and at a temperature below 40° C. to obtain 8 kilos of a blandfluid syrup. The previous extract is solubilised in a 100 litre mix ofethanol: water (60:40) at 50° C. by stirring for 2 hours and it ispurified by filtering through a 5 micrometers membrane. The filtrate isof reduced concentration and obtains a hot fluid oleoresin that changesto paste form when cooled. This obtains 7.5 kilos of Hypericumperforatum L oleoresin with a hyperforin content of 14% and a hypericinecontent of 0.6%.

EXAMPLE 2 Illustrating the Stability of the Extracts

[0031] 1.—Stability at Room Temperature

[0032] T=0 % Hyperforin Adhyperforin Total Hyperforins Extract A 7.7%7.3%   15% Extract B 7.1% 6.7% 13.8% Extract C 7.7% 7.2% 14.9% Extract D0.6% 0.4%   1% T = 12 months at 25° C. Extract A 7.4% 7.2%   14% ExtractB 8.6% 7.0% 15.6% Extract C 7.2% 7.5% 14.9% Extract D 0.1% 0.1%  0.2%

[0033] Where extracts A, B and C are Hypericum perforatum L oleoresinsobtained according to the invention and extract D is a Hypericumperforatum L oleoresin obtained by extraction with ethanol, water(50:50).

[0034] The content in hyperforin was determined by HPLC.

[0035] 2.—Stability at 40° C.

[0036] T=0 Hyperforin Adhyperforin Total Hyperforins 2. - Stability at40° C. T = 0 7.7% 7.,3%   15% T = 12 months at 40° C. 7.2% 6.9% 14.1%3. - Stability in the presence of light T = 0 7.7% 7.3%   15% T = 12Subjected to natural light   9% 6.8% 15.8%

[0037] 4.—Conclusion

[0038] Hypericum perforatum L oleoresins obtained according to theinvention are stable and the hyperforin content is not decreased witheither temperature or light.

EXAMPLE 3 Illustrating the Regulation of the Production of theComponents of the Extracellular Membrane

[0039] Cellular Cultures

[0040] The fibroblasts were obtained from surgical material. The skinsamples were pre-incubated for 2 hours at 40 °C. in RPMI 1640 with 2%penicillin/streptomycin. The fatty tissues were eliminated and the skinwas cut into small pieces and fixed to culture dishes dampened withfoetal calf serum (FCS). The pieces of skin were incubated at 37° C. ina CO₂ atmosphere in RPMI with 10% FCS and 1% penicillin/streptomycin.The culture medium was changed twice a week. The fibroblast culture wastrypsinised (trypsin/EDTA: 0.0%/0.02%) and a subculture was started,using the cells from the ₄ ^(th) to the 14th

[0041] Collagen Synthesis

[0042] The human fibroblasts were grown in microplates for cell tissues.Each plate was inoculated with 10,000 cells in 100 μl of RMPI mediumsupplemented with FCS (10%) and ascorbic acid (50 μl/ml). After 24 hoursof incubation in a humid CO₂ atmosphere at 5%, the culture medium waschanged for 100 μl of fresh medium per plate containing differentconcentrations of the extract to be analysed and 1μ Ci ³H of markedproline. After 24 hours of incubation, the collagen was extracted fromeach plate by adding 100 μl of 1M acetic acid that contained 1 mg/ml ofpepsin and stored at 4° C. throughout the night. The content of theplates was transferred to polypropylene tubes to which 800 μl of 0.5Macetic acid containing a neutral soluble rat skin collagen salt (200 μl)as a dilutant, was added. The tubes were centrifuged at 4000 g for 20minutes. The collagen was precipitated from the supernatant by adding250 μl of NaCl in acetic acid (25%) per tube. After 2 hours, the tubeswere centrifuged at 4000 g for 30 minutes, the precipitates werere-dissolved in 300 μl 0.15 M NaCl in 0.05 M tris-HCl, pH 7.5. Thecollagen was precipitated by adding 2 ml of 4.5 M NaCl in the samebuffer. After 2 hours, the tubes were centrifuged at 4000 g for 30minutes. The supernatant was discarded and the collagen precipitateswere washed in 2 ml of 2% ethanol and centrifuged at 4000 g for 30minutes. Finally, each precipitate was dissolved in 250 μl of aceticacid 0.5 M, taken to a scintillation vial and measured in a liquidscintillation counter with an external standard.

[0043] The percentage of incorporation of collagen for differentconcentrations of oleoresin are showed as follows. The value indicatedis the average from 4 parallel experiments.

[0044] % of incorporation of H³-proline in the collagen. CONTROL 100%0.1% Ethanol 126% 0.01 μg/ml 120% 0.1 μg/ml 115% 0.5 μg/ml 130% 1 μg/ml170% 5 μg/ml 20% 10 μg/ml 10% 50 μg/ml 20%

[0045] Tenascine

[0046] The fibroblasts were grown on microplates with a density of20,000 cells per plate in RPMI medium without FCS. After 24 hours at 37°C. in a 5% CO₂ atmosphere, the culture medium was changed. The cellswere incubated for 48 hours more at 37° C. with different concentrationsof oleoresin. The cells were washed three times with PBS with 1% BSA and0.1% Tween 20. The cells were fixed with a methanol/acetone solution(1:1), washed 3 times as described above, and incubated with amonoclonal anti-tenascine antibody for 1 hour at 37° C. After washing,the cells were incubated with a monoclonal anti-mouse goat antibody withalkaline phosphatase. The cells were washed 3 times and incubated withp-nitrophenyl phosphate (1 mg/ml) for 15 minutes. The microplates werecentrifuged at 200 g and 100 μl of the supernatant were transferred to anew microplate. The plates were read in an ELISA reader at 405 nm.

[0047] The percentage of the tenascine content compared to the controlfor different concentrations of oleoresins is shown as follows, wherethe values are the average from 3 experiments.

[0048] % de tenascine compared to the control. CONTROL 100% 0.1% Ethanol115% 0.1 μg/ml 90% 0.5 μg/ml 60% 1 μg/ml 50% 5 μg/ml 40% 10 μg/ml 30% 20μg/ml 20%

EXAMPLE 4 Illustrating the Results Obtained by the HydrosolubleHypericum Perforatum L Oleoresin Gel in the Cicatrizant

[0049] Product A.—Hydrosoluble gel with a content of 0.1% of Hypericumperforatum L oleoresin.

[0050] Product B.—Hydrosoluble gel with a content of 0.5% of Hypericumperforatum L oleoresin.

[0051] Group 1.—12 patients with dermatological pathology requiringremoval by electric scalpel, liquid nitrogen or laser, with no need forsutures. The burns are treated clinically.

[0052] Group 2.—Patients with dermatological disease requiring removaland surgical closure with sutures. Wounds treated clinically.

[0053] Group 1 results 1: Surgical burns.

[0054] 48-hour control. Group 1 results 1: Surgical burns. Pain ErythemaInfection Crust 48-hour control. Product A Slight Slight No Yes ProductB Moderate Slight Slight Yes 7-day control. Product A No No No HGTProduct B No No No GNT

[0055] Group 2 results: surgical wounds. Pain Erythema Infection CrustSuture 48-hour control. Product Moderate Moderate Slight Ini- Normal Atial Product Moderate Moderate Moderate No Normal B 7-day controlProduct No No No Yes Re- A moved Product No No No Yes Re- B moved 15-daycontrol. No hypertrophic or cheloid scars were observed. Conclusions.Cicatrizant effect The products have been well tolerated and nei- therinflammation or infection has appeared. There are no collateral effectsThere is no hypertrophic scarring The lesions are easily monitoredwithout remov- ing the product. No maceration of the skin was observed.

1. Hypericum perforatum L oleoresin containing no less than 10% ofhyperforin and no less than 0.5% of hypericines, characterised in thatit does not contain preservatives and the hyperforin does not decreaseover time.
 2. Procedure for preparing a Hypericum perforatum L oleoresinwith a stable hyperforin content, characterised in that it consist of:a) Extraction of the Hypericum perforatum L with one or several organicsolvents with polarity less than 0.6. b) Evaporation of the solvent. c)Dissolution of the primary extract obtained in the previous steps withalcanol-water mixes at 40-50° C., followed by filtration. d) Evaporationof the solvent at low pressure.
 3. Procedure for obtaining an oleoresinin accordance with claim 2, characterised in that the alcanols used inthe purification of the extract are methanol, ethanol or isopropanol. 4.Procedure for obtaining a Hypericum perforatum L resin in accordancewith claims 2 and 3, characterised in that the alcanol mixes areethanol:water (60:40)
 5. Procedure for obtaining a Hypericum perforatumL oleoresin in accordance with any of claims 2, 3 and 4, characterisedin that the plant is dried at under 35° C.
 6. Procedure for obtaining aHypericum perforatum L oleoresin in accordance with any of claims 2, 3,4, 5 and 6, characterised in that the content in hyperforins in theplant is over 2%.
 7. Use of a Hypericum perforatum oleoresin inaccordance with any of claims 1 to 6, to prepare a drug to regulate theproduction of the components of the extracellular matrix in humanfibroblasts.
 8. Use of a Hypericum oleoresin in accordance with any ofclaims 1 to 6, to manufacture a drug to regulate collagen production. 9.Use of a Hypericum oleoresin in accordance with any of claims 1 to 6 tomanufacture a drug to regulate tenascine production.
 10. Hydrosolublegel usable as a cicatrizant, characterised in that it contains as anactive ingredient a Hypericum perforatum L oleoresin, dilutant agents,gel formers, emulsifiers and preservatives.
 11. Hydrosoluble gel inaccordance with claim 10, characterised in that the dilutant is water.12. Hydrosoluble gel in accordance with claim 11, characterised in thatthe gelifying agent is glyceril polyacrilate.
 13. Hydrosoluble gel inaccordance with claim 11, characterised in that the humidifying agent isglycerine.
 14. Hydrosoluble gel in accordance with claim 11,characterised in that the emulsifying agents are PEG 40-hydrogenatedcastor oil, polysorbate-20 and/or octoxinole-11.
 15. Hydrosoluble gel inaccordance with claim 11, characterised in that the preservative agentsare parabenes.
 16. Procedure for obtaining a hydrosoluble gel inaccordance with claims 10 to 15, characterised in that it consists of:a) Dissolution of the Hypericum perforatum L oleoresin in theemulsifiers. b) Dissolution of the preservatives in water. c)Incorporation of the mixture of the Hypericum oleoresin and theemulsifiers in the preservative solution. d) Incorporation into theprevious mix of the humidifier and the gelifying agent, with stirring.